Summary Antibiotic resistance is an acute problem in US hospitals, and threatens every branch of medicine as currently practiced. Antibiotic resistance is now even a national security concern. Staphylococcus aureus (esp. MRSA) and the enterococci (esp. VRE) are among top causes of hospital infection that are especially difficult to treat, because of multiple drug resistances and intrinsic hardiness. This proposal, in collaboration and synergy with all other subprojects, attempts to respond to the national challenge by building on advances made in the previous period of support, and focusing those resources sharply on advancing tangible solutions to this crisis. Our overarching goals are to improve the utility of antibiotic classes that currently exist, and to explore new therapeutic paradigms for multidrug resistant infection. To achieve this goal, in this Subproject, we will pursue 2 Specific Aims: 1) Identify impediments in MRSA/MSSA and VRE/VSE to antibiotic activity, and 2) Explore a new paradigm for VRE infection prevention and treatment ? Gut ecology management. This project takes advantage of cutting-edge technologies and unique assets, including a crowd sourced strain collection, to obtain new information from which new and better approaches for preventing and treating multidrug resistant MRSA and VRE infections can be designed. We have pioneered the application of comparative genomics to understand the origin and spread of antibiotic resistances among enterococci; showing in the previous period, that anthropogenic factors such as the urbanization of humans and the application of antibiotics in unprecedented levels, has driven their evolution; and in collaboration with other subprojects, applied Tn-seq, to identify genes required for S. aureus growth in vitro and in vivo as well as new drug targets. Each has been precedent-setting in application to staphylococci and enterococci. These technologies now will be used to identify impediments to target inhibition by antibiotics, and to inform the design of new antibiotics and antibiotic potentiators. Further, a new treatment paradigm will be explored for prevention of VRE colonization and infection. These results will provide critical information for optimizing the design of new drugs and antibiotic potentiators; and new tools for improving the ecological management of patients to reduce the likelihood and numbers of multidrug resistant infections. We believe this data will be of substantial value in directly addressing the antibiotic resistance crisis that now exists.